The invention relates to the field of sequencing of polynucleotides and/or proteins as well as fragments of such compounds. More particularly, it relates to methods for analyzing their structure and molecular weights.
There is an increasing demand for reliable and inexpensive methods for the sequencing of polynucleotides, such as, DNA, RNA and the like. Generally, a radioactive or fluorescent probe, which selectively hybridizes to a specific target nucleic acid, is added to the support. A typical common type of probe is a single-stranded (ss) DNA which is complementary to a sequence in the target DNA or RNA.
The hybrid molecule thus formed with the label probe thereon may then be detected by various techniques depending on the nature of the label used. An example of such hybridization is shown in U.S. Pat. No. 4,358,535.
Typical labeling probes include the incorporation of a radioactive atom, such as, .sup.32 p, .sup.14 C, or .sup.3 H. This can be achieved by nick translation, such as that shown in Rigbny et al (J. Mol. Biol., 113: 237, 1977), wherein a labeled nucleotide is incorporated into a gap created in the DNA of the probe. Other labels can be introduced by nick translation, for example, by incorporating biotinylated nucleosides which can then be coupled to an avidin bound label, such as, an enzyme. The DNA can also be labeled with antigenic groups reacting with antibodies.
For the assay or quantification of nucleic acids, such as, DNA or mRNA, either the total nucleic acid material present in the sample or that transcribed from a specific gene can be conventionally determined by this so-called dot-blot analysis technique.
One of the problems with such sequencing techniques requires the handling of radioactive isotopes and presents an environmentally undesirable situation in the laboratory. The use of fluorescent labels or enzyme labels results in relatively complicated techniques for the ultimate reading of the label. Generally, expensive equipment and relatively skilled technicians are needed to effect the analysis of gels and/or substrates onto which the labeled molecules or segments have been fixed.
In addition, numerous attempts have been made to automate the electrophoresis step, detection and data handling. However, difficulties have arisen with respect to such methods primarily because of the method of labeling.
The storage and reading of material in magnetic media is widespread with the use of floppy drives, hard drives, and digital and analog tapes. Superconducting quantum interference devices (SQUID) and magnetic force microscopes (MFM) have been in use to measure magnetic fields on microscopic levels. These systems, however, are extremely costly. In addition, both SQUID and MFM devices are limited to the measurement of magnetic fields in materials with extremely smooth surfaces.